Detection of Proteins on Blot Membranes

Aaron Goldman1, Sandra Harper1, David W. Speicher1

1 The Wistar Institute, Philadelphia, Pennsylvania
Publication Name:  Current Protocols in Protein Science
Unit Number:  Unit 10.8
DOI:  10.1002/cpps.15
Online Posting Date:  November, 2016
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Abstract

Staining of blot membranes enables the visualization of bound proteins. Proteins are usually transferred to blot membranes by electroblotting, by direct spotting of protein solutions, or by contact blots. Staining allows the efficiency of transfer to the membrane to be monitored. This unit describes protocols for staining proteins after electroblotting from polyacrylamide gels to blot membranes such as polyvinylidene difluoride (PVDF), nitrocellulose, or nylon membranes. The same methods can be used if proteins are directly spotted, either manually or using robotics. Protocols are included for seven general protein stains (amido black, Coomassie blue, Ponceau S, colloidal gold, colloidal silver, India ink, and MemCode) and three fluorescent protein stains (fluorescamine, IAEDANS, and SYPRO Ruby). Also included is an in‐depth discussion of the different blot membrane types and the compatibility of different protein stains with downstream applications, such as immunoblotting or N‐terminal Edman sequencing. © 2016 by John Wiley & Sons, Inc.

Keywords: electroblots; electrotransfer efficiency; membrane stain; protein stain

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Amido Black Staining
  • Basic Protocol 2: Coomassie Blue R‐250 Staining
  • Basic Protocol 3: Ponceau S Staining
  • Basic Protocol 4: Colloidal Gold Staining
  • Basic Protocol 5: Colloidal Silver Staining
  • Basic Protocol 6: India Ink Staining
  • Basic Protocol 7: MemCode Staining
  • Basic Protocol 8: Fluorescamine Labeling
  • Alternate Protocol 1: IAEDANS Labeling
  • Alternate Protocol 2: SYPRO Ruby Labeling
  • Commentary
  • Literature Cited
  • Tables
     
 
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Materials

Basic Protocol 1: Amido Black Staining

  Materials
  • Blot transfer membrane (unit 10.7; Goldman et al., )
  • Coomassie blue stain: 0.025% (w/v) Coomassie brilliant blue R‐250 (e.g., Bio‐Rad, Sigma‐Aldrich, ThermoFisher Scientific, etc.) in 40% methanol/7% acetic acid (v/v)
  • 50% methanol/7% acetic acid (v/v)
  • Plastic box

Basic Protocol 2: Coomassie Blue R‐250 Staining

  Materials
  • Blot transfer membrane (unit 10.7; Goldman et al., )
  • Ponceau S stain: 0.5% (w/v) Ponceau S (e.g., Sigma‐Aldrich, ThermoFisher Scientific) in 1% (v/v) acetic acid
  • 200 µM NaOH/20% (v/v) acetonitrile
  • Plastic box

Basic Protocol 3: Ponceau S Staining

  Materials
  • Blot transfer membrane (unit 10.7; Goldman et al., )
  • Tween 20 solution: 0.3% (v/v) Tween 20 in PBS ( appendix 2E; prepare solution fresh weekly and store at 4°C)
  • Colloidal gold reagent (e.g., Bio‐Rad)
  • Glass box

Basic Protocol 4: Colloidal Gold Staining

  Materials
  • Blot transfer membrane (unit 10.7; Goldman et al., )
  • 40% (w/v) sodium citrate (store at 4°C for several months)
  • 20% (w/v) ferrous sulfate (FeSO 4⋅7H 2O), prepared fresh
  • 20% (w/v) silver nitrate (store at 4°C for several months)
  • Glass box

Basic Protocol 5: Colloidal Silver Staining

  Materials
  • Blot transfer membrane (unit 10.7; Goldman et al., )
  • Tween 20 solution: 0.3% (v/v) Tween 20 in PBS ( appendix 2E; prepare solution fresh weekly and store at 4°C)
  • India ink solution: 0.1% (v/v) India ink (Pelikan 17 black) in Tween 20 solution (store 1 month at room temperature)
  • Plastic box

Basic Protocol 6: India Ink Staining

  Materials
  • Blot transfer membrane (unit 10.7; Goldman et al., )
  • MemCode Reversible Protein Stain Kit for PVDF or nitrocellulose membranes (ThermoFisher Scientific/Pierce) including:
    • MemCode Sensitizer
    • MemCode Reversible Protein Stain
    • MemCode Destain
    • MemCode Stain Eraser
  • Destain/methanol solution (1:1 mixture of reagent‐grade methanol and MemCode Destain)
  • Eraser/methanol solution (1:1 mixture of reagent‐grade methanol and MemCode Eraser)
  • Plastic box
  • Rotary shaker

Basic Protocol 7: MemCode Staining

  Materials
  • Blot transfer membrane (unit 10.7; Goldman et al., )
  • Sodium bicarbonate solution: 100 mM sodium bicarbonate in 0.3% (v/v) Tween 20, pH 9.0 (prepare fresh weekly and store at 4°C)
  • Fluorescamine stain: 0.25 mg/ml fluorescamine (Sigma‐Aldrich) in sodium bicarbonate solution (prepare fresh daily)
  • Plastic box

Basic Protocol 8: Fluorescamine Labeling

  Materials
  • Blot transfer membrane (unit 10.7; Goldman et al., )
  • DTT solution: 200 mM dithiothreitol (DTT) in 100 mM Tris⋅Cl, pH 8.6 ( appendix 2E; prepare immediately before use)
  • 100 mM Tris⋅Cl, pH 8.6 ( appendix 2E)
  • N‐iodoacetyl‐N′‐(5‐sulfo‐1‐naphthyl)ethylenediamine (IAEDANS; Sigma‐Aldrich, ThermoFisher Scientific, etc.; store desiccated in the dark at −20°C)
  • Glass box
  • UV lamp

Alternate Protocol 1: IAEDANS Labeling

  Materials
  • Blot transfer membrane (unit 10.7; Goldman et al., )
  • 7% acetic acid/10% reagent grade methanol
  • SYPRO Ruby protein blot stain (Life Technologies)
  • Plastic box
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Figures

Videos

Literature Cited

  Antharavall, B.S., Carter, B., Bell, P.A., and Mallia, A.K. 2004. A high‐affinity reversible protein stain for Western blots. Anal. Biochem. 329:276‐280. doi: 10.1016/j.ab.2004.02.049.
  Berggren, K., Steinberg, T.H., Lauber, W.M., Carroll, J.A., Lopez, M.F., Chernokalskaya, E., Zieske, L., Diwu, Z., Haugland, R.P., and Patton, W.F. 1999. A luminescent ruthenium complex for ultrasensitive detection of proteins immobilized on membrane supports. Anal. Biochem. 276:129‐143. doi: 10.1006/abio.1999.4364.
  Goldman, A., Ursitti, J.A., Mozdzanowski, J., and Speicher, D.W. 2015. Electroblotting from polyacrylamide gels. Curr. Protoc. Protein Sci. 82:10.7.1‐10.7.16. doi: 10.1002/0471140864.ps1007s82.
  Hancock, K. and Tsang, V.C.M. 1983. India ink staining of proteins on nitrocellulose paper. Anal. Biochem. 133:157‐162. doi: 10.1016/0003‐2697(83)90237‐3.
  Kikuchi, S., Bedard, J., and Nakai, M. 2011. One‐ and two‐dimensional blue native‐page and immunodetection of low‐abundance chloroplast membrane protein complexes. Methods Mol. Biol. 775:3‐17. doi: 10.1007/978‐1‐61779‐237‐3_1.
  Lee, D.Y. and Chang, G.D. 2015. Post‐staining electrophoresis for efficient and reliable peptide blotting. Methods Mol. Biol. 1312:185‐195 doi: 10.1007/978‐1‐4939‐2694‐7_22.
  Moeremans, M., Daneels, G., and De Mey, J. 1985. Sensitive colloidal metal (gold or silver) staining of protein blots on nitrocellulose membranes. Anal. Biochem. 145:315‐321. doi: 10.1016/0003‐2697(85)90368‐9.
  Mozdzanowski, J. and Speicher, D.W. 1992. Microsequence analysis of electroblotted proteins. Anal. Biochem. 207:11‐18. doi: 10.1016/0003‐2697(92)90492‐P.
  Park, J., Mabuchi, M., and Sharma, A. 2015. Visualization of unstained protein bands on PVDF. Methods Mol. Biol. 1314:191‐194. doi: 10.1007/978‐1‐4939‐2718‐0_21.
  Reig, J. and Klein, D. 1988. Submicrogram quantities of unstained proteins are visualized on polyvinylidene difluoride membranes by transillumination. Appl. Theor. Electrophor. 1:59‐60.
  Shimazaki, Y. and Michhiro, M. 2013. Analysis of trypsin inhibition activity in human plasma proteins after separation by non‐denaturing two‐dimensional electrophoresis. Clin. Chim. Acta 425:48‐53. doi: 10.1016/j.cca.2013.07.004.
  Speicher, D.W. 1989. Microsequencing with PVDF membranes: Efficient electroblotting, direct protein adsorption and sequencer program modifications. In Techniques in Protein Chemistry (T.E. Hugli, ed.) pp. 24‐35. Academic Press, San Diego.
  Sutherland, M.W. and Skerritt, J.H. 1986. Alkali enhancement of protein staining on nitrocellulose. Electrophoresis 7:401‐406. doi: 10.1002/elps.1150070903.
  Vera, J.C. and Rivas, C. 1988. Fluorescent labeling of nitrocellulose‐bound proteins at the nanogram level without changes in immunoreactivity. Anal. Biochem. 173:399‐404. doi: 10.1016/0003‐2697(88)90206‐0.
Key References
  Moeremans et al., 1985. See above.
  Describes a method for preparation of colloidal metal stains.
  Vera, J.C. and Rivas, C. 1988. See above.
  Describes the use of multiple detection methods.
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